Clinical failure of total hip replacements involving polyethylene cups and cobalt-chrome femoral components will affect more than a million individuals in the coming 20-year period. Failure in most of these implants will result from the release of polyethylene (PE) wear debris, the appearance of PE particle-dependent inflammation and consequent, aseptic loosening of one or both of the components of the hip prosthesis. The overall aim of this proposal is 1] to ascertain the means by which the exfoliated PE particles engage cells of the host to initiate the innate immune response and 2] to identify opportunities for targeted therapeutic, and perhaps inexpensive, intervention in periprosthetic osteolytic events. As such, it is hypothesized that PE wear particles shed locally by the implant activate Toll-like receptors (TLRs), a subset of pattern recognition receptors embedded in the plasma membrane of macrophages, to direct the elaboration of inflammatory cytokines responsible for osteolysis (e.g. RANKL). This proposal is made possible by the recent discoveries that 1] one can now isolate, purify and characterize in high yield the full spectrum of PE particles generated during physiologic wear simulation and 2] innate immune signaling in primary isolates of human macrophages is mediated by TLRs and impaired by a vitamin D-insufficient status in the host. Two aims are proposed to test the above-stated hypothesis. Specific Aim 1 will seek to determine the TLR response to exposure to authentic, pure wear particles derived from hip simulator studies of conventional polyethylene acetabular cups against cobalt-chrome femoral balls in terms of monokine gene and protein expression. Here TLR-expressing mouse bone marrow-derived macrophages from wild-type and from TLR- and MyD88 (universal adapter protein for TLRs)-deficient mice will be employed to monitor by quantitative PCR and/or ELISA expression of the TLRs and monokines which are known to mediate bone resorption. Initial screens for involved TLRs and TLR-TLR cooperation will utilize TLR-targeted lentiviral shRNAmir expression for loss-of-function RNA interference. Specific Aim 2 will attempt to ascertain in vivo in the mouse subcutaneous air pouch model whether the vitamin D system influences TLR responsivity to wear particles in terms of 1] vitamin D metabolite response modifiers (e.g. the vitamin D-hydroxylases and vitamin D receptor [VDR]), 2] TLR expression and 3] cytokine responses to particle stimulation. These markers of bioresponse will be analyzed quantitatively in vivo under conditions of vitamin D sufficiency, insufficiency, rescue from insufficiency, and TLR and MyD88 deficiency. Considering that the aging population in this country, the majority of which are vitamin D- insufficient, encompasses those who i) already harbor a failing prosthesis or ii) are candidates to receive a hip implant, then reversal of the vitamin D-insufficient state may represent a simple, cost-efficient and effective means of improving outcomes with hip implants in particular and in joint arthroplasties in general. PUBLIC HEALTH RELEVANCE: Clinical failure of existing total hip replacements, estimated to occur in more than a million individuals in the coming 20-year period, results from the release of polyethylene (PE) wear debris from the implant, PE particle- dependent inflammation and consequent periprosthetic osteolysis and loosening of the components of the prosthesis. It is hypothesized that PE wear particles shed locally by the implant will activate the Toll-like receptor (TLR), vitamin D-dependent signaling pathway in macrophages to direct the elaboration of inflammatory cytokines responsible for osteolysis (e.g. RANKL). Considering that the aging population in this country, the majority of which are vitamin D-insufficient, encompasses those who i) already harbor a failing prosthesis or ii) are candidates to receive a hip implant, then reversal of the vitamin D-insufficient state may represent a simple, cost-efficient and effective means of improving outcomes with hip implants in particular and in joint arthroplasties in general.